Interpretive Summary: With the increasing pressures against, and the increasing number of restrictions on animal research, it is important that, wherever possible, alternatives to animal experimentation be developed. In nutritional research, the use of cultures of cells that mimic the intestinal lining is an alternative to the use of animals for experiments examining nutrient absorption. CACO-2 cells are such a cell type; they have been utilized to study the absorption of many nutrients, including trace elements. However, with any cell line, the way in which the cells are grown can affect the results. In this report, we have examined several aspects of CACO-2 cell culture conditions and discussed how they may influence the results of nutritional experiments. We have shown that, as the cell line ages, its metabolism and growth characteristics change. We have also shown that the cell line will spontaneously form multiple layers, and these layers may change parameters associated with absorption. By helping define optimal experimental conditions for Caco-2 cells, this study has improved the value of these cells as a model of the intestinal epithelium and as an in vitro alternative to animal experimentation.

Technical Abstract:
The Caco-2 cell line is used by many investigators as a model of the intestinal epithelium to study nutrient uptake and transport. We have examined several problems associated with Caco-2 cells that may influence their suitability as a model. To study the influence of passage on the model, cultures were monitored from passage 17 to 109. Transepithelial electrical resistance (TEER) and sucrase activity (measured in 21-day-old cultures) increased through about passage 35. TEER values declined after about passage 60, sucrase remained elevated but variable. Cells previously frozen at passage 22, 33 and 72 were thawed and grown simultaneously for 24 days. Older-passaged cells grew faster initially. Before day 15, passage 72 cells had higher TEER and lower permeability to 14C-mannitol than passages 22 and 33; however, after day 15 all passages showed similar permeability. On day 21, passage 72 cells had significantly lower alkaline phosphatase activity than did the other passages. Electron microscopy did not reveal any major morphological differences between the passages; however, it did show that some areas of cells grown on membranes were not monolayers, but were several cells thick with varied morphology. Investigation of the formation of these multilayered areas showed them to be an inherent part of cell growth under the conditions used. These results emphasize the inherent variability in Caco-2 cell models, and emphasize the need to closely monitor the culture characteristics during growth and differentiation under specific experimental conditions.